Humans grow corn for food and energy applications. Corn is an important crop. It is an important source of food, food products, and animal feed in many areas of the world. Insects eat and damage plants and thereby undermine these human efforts. Billions of dollars are spent each year to control insect pests and additional billions are lost to the damage they inflict.
Damage caused by insect pests is a major factor in the loss of the world's corn crops, despite the use of protective measures such as chemical pesticides. In view of this, insect resistance has been genetically engineered into crops such as corn in order to control insect damage and to reduce the need for traditional chemical pesticides.
Over 10 million acres of U.S. corn are infested with corn rootworm species complex each year. The corn rootworm species complex includes the northern corn rootworm (Diabrotica barberi), the southern corn rootworm (D. undecimpunctata howardi), and the western corn rootworm (D. virgifera virgifera). (Other species include Diabrotica virgifera zeae (Mexican corn rootworm), Diabrotica balteata (Brazilian corn rootworm), and Brazilian corn rootworm complex (Diabrotica viridula and Diabrotica speciosa).)
The soil-dwelling larvae of these Diabrotica species feed on the root of the corn plant, causing lodging. Lodging eventually reduces corn yield and often results in death of the plant. By feeding on cornsilks, the adult beetles reduce pollination and, therefore, detrimentally affect the yield of corn per plant. In addition, adults and larvae of the genus Diabrotica attack cucurbit crops (cucumbers, melons, squash, etc.) and many vegetable and field crops in commercial production as well as those being grown in home gardens.
Synthetic organic chemical insecticides have been the primary tools used to control insect pests but biological insecticides, such as the insecticidal proteins derived from Bacillus thuringiensis (Bt), have played an important role in some areas. The ability to produce insect-resistant plants through transformation with Bt insecticidal protein genes has revolutionized modern agriculture and heightened the importance and value of insecticidal proteins and their genes.
Insecticidal crystal proteins from some strains of Bacillus thuringiensis (B.t.) are well-known in the art. See, e.g., Hofte et al., Microbial Reviews, Vol. 53, No. 2, pp. 242-255 (1989). These proteins are typically produced by the bacteria as approximately 130 kDa protoxins that are then cleaved by proteases in the insect midgut, after ingestion by the insect, to yield a roughly 60 kDa core toxin. These proteins are known as crystal proteins because distinct crystalline inclusions can be observed with spores in some strains of B.t. These crystalline inclusions are often composed of several distinct proteins.
One group of genes which have been utilized for the production of transgenic insect resistant crops are the delta-endotoxins from Bacillus thuringiensis (B.t.). Delta-endotoxins have been successfully expressed in crop plants such as cotton, potatoes, rice, sunflower, as well as corn, and have proven to provide excellent control over insect pests. (Perlak, F. J et al. (1990) Bio/Technology 8, 939-943; Perlak, F. J. et al. (1993) Plant Mol. Biol. 22: 313-321; Fujimoto H. et al. (1993) Bio/Technology 11: 1151-1155; Tu et al. (2000) Nature Biotechnology 18:1101-1104; PCT publication number WO 01/13731; and Bing J W et al. (2000) Efficacy of Cry1F Transgenic Maize, 14th Biennial International Plant Resistance to Insects Workshop, Fort Collins, Colo.)
Several Bt proteins have been used to create the insect-resistant transgenic plants that have been successfully registered and commercialized to date. These include Cry1Ab, Cry1Ac, Cry1F, Cry1A.105, Cry2Ab, Cry3Aa, Cry3Bb, and Cry34/35Ab in corn, Cry1Ac and Cry2Ab in cotton, and Cry3A in potato.
The commercial products expressing these proteins express a single protein except in cases where the combined insecticidal spectrum of 2 proteins is desired (e.g., Cry1Ab and Cry3Bb in corn combined to provide resistance to lepidopteran pests and rootworm, respectively) or where the independent action of the proteins makes them useful as a tool for delaying the development of resistance in susceptible insect populations (e.g., Cry1Ac and Cry2Ab in cotton combined to provide resistance management for tobacco budworm).
Some of the qualities of insect-resistant transgenic plants that have led to rapid and widespread adoption of this technology also give rise to the concern that pest populations will develop resistance to the insecticidal proteins produced by these plants. Several strategies have been suggested for preserving the utility of Bt-based insect resistance traits which include deploying proteins at a high dose in combination with a refuge, and alternation with, or co-deployment of, different toxins (McGaughey et al. (1998), “B.t. Resistance Management,” Nature Biotechnol. 16:144-146).
The proteins selected for use in an Insect Resistance Management (IRM) stack should be active such that resistance developed to one protein does not confer resistance to the second protein (i.e., there is not cross resistance to the proteins). If, for example, a pest population selected for resistance to “Protein A” is sensitive to “Protein B”, one would conclude that there is not cross resistance and that a combination of Protein A and Protein B would be effective in delaying resistance to Protein A alone.
In the absence of resistant insect populations, assessments can be made based on other characteristics presumed to be related to cross-resistance potential. The utility of receptor-mediated binding in identifying insecticidal proteins likely to not exhibit cross resistance has been suggested (van Mellaert et al. 1999). The key predictor of lack of cross resistance inherent in this approach is that the insecticidal proteins do not compete for receptors in a sensitive insect species.
In the event that two Bt toxins compete for the same receptor, then if that receptor mutates in that insect so that one of the toxins no longer binds to that receptor and thus is no longer insecticidal against the insect, it might be the case that the insect will also be resistant to the second toxin (which competitively bound to the same receptor). That is, the insect is said to be cross-resistant to both Bt toxins. However, if two toxins bind to two different receptors, this could be an indication that the insect would not be simultaneously resistant to those two toxins.
A relatively newer insecticidal protein system was discovered in Bacillus thuringiensis as disclosed in WO 97/40162. This system comprises two proteins—one of approximately 14-15 kDa and the other of about 44-45 kDa. See also U.S. Pat. Nos. 6,083,499 and 6,127,180. These proteins have now been assigned to their own classes, and accordingly received the Cry designations of Cry34 and Cry35, respectively. See Crickmore et al. website (biols.susx.ac.uk/home/Neil_Crickmore/Bt/). Many other related proteins of this type of system have now been disclosed. See e.g. U.S. Pat. No. 6,372,480; WO 01/14417; and WO 00/66742. Plant-optimized genes that encode such proteins, wherein the genes are engineered to use codons for optimized expression in plants, have also been disclosed. See e.g. U.S. Pat. No. 6,218,188.
The exact mode of action of the Cry34/35 system has yet to be determined, but it appears to form pores in membranes of insect gut cells. See Moellenbeck et al., Nature Biotechnology, vol. 19, p. 668 (July 2001); Masson et al., Biochemistry, 43 (12349-12357) (2004). The exact mechanism of action remains unclear despite 3D atomic coordinates and crystal structures being known for a Cry34 and a Cry35 protein. See U.S. Pat. Nos. 7,524,810 and 7,309,785. For example, it is unclear if one or both of these proteins bind a typical type of receptor, such as an alkaline phosphatase or an aminopeptidase.
Furthermore, because there are different mechanisms by which an insect can develop resistance to a Cry protein (such as by altered glycosylation of the receptor [see Jurat-Fuentes et al. (2002) 68 AEM 5711-5717], by removal of the receptor protein [see Lee et al. (1995) 61 AEM 3836-3842], by mutating the receptor, or by other mechanisms [see Heckel et al., J. Inv. Pathol. 95 (2007) 192-197]), it was impossible to a priori predict whether there would be cross-resistance between Cry34/35 and other Cry proteins. Lefko et al. discusses a complex resistance phenomenon in rootworm. J. Appl. Entomol. 132 (2008) 189-204.
Predicting competitive binding for the Cry34/35 system is also further complicated by the fact that two proteins are involved in the Cry34/35 binary system. Again, it is unclear if and how these proteins effectively bind the insect gut/gut cells, and if and how they interact with or bind with each other.
Other options for controlling coleopterans include Cry3Bb toxins, Cry3C, Cry6B, ET29, ET33 with ET34, TIC407, TIC435, TIC417, TIC901, TIC1201, ET29 with TIC810, ET70, ET76 with ET80, TIC851, and others. RNAi approaches have also been proposed. See e.g. Baum et al., Nature Biotechnology, vol. 25, no. 11 (November 2007) pp. 1322-1326.
Meihls et al. suggest the use of refuges for resistance management in corn rootworm. PNAS (2008) vol. 105, no. 49, 19177-19182.